Skin formulation, preparation and uses thereof

ABSTRACT

Serum compositions for application to skin are described which contain an amniotic fluid extract in combination with embryonic stem cells. Formulations containing the serum composition are also described. The serum compositions and formulations may be applied to skin for treatment of symptoms of aging, wounds, burns, scars or other skin lesions. Methods of preparing the serum compositions and formulations are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/524,941, filed Aug. 18, 2011, which is incorporated herein byreference.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

Not applicable

PARTIES OF JOINT RESEARCH AGREEMENT

Not applicable

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

Not applicable

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to compositions containing embryonicstem cells and extracellular fluid which may be applied to the skin toobtain beneficial results including a decrease in the appearance ofaging of the skin, and treatment for healing wounds, burns and scars.

2. Description of the Related Art

Skin is the largest organ of the human body. Not only does this 1.8square meter network of nerves, blood vessels, pigments, and fibrouscells as well as sweat and oil glands play a key role in protecting thebody against pathogens, but skin also prevents excessive water loss.Skin also has other important functions such as insulation, temperaturehomeostasis, sensation, synthesis of vitamin D, and the protection ofvitamin B. Skin care, especially anti-aging care products, focus on oneor more of the following aspects: antioxidant protection, moistureretention, and/or promoting skin cell growth.

Many synthetic chemicals as well as natural extracts have beenidentified as effective ingredients for skin care. However, there arealso misunderstandings about the function of these ingredients. Many“anti-aging” products are, in fact, make-up products using compoundssuch as silicon to cover up lines and wrinkles. Natural ingredients aregenerally gentler to skin than synthetic chemicals with fewer unwelcomeeffects.

The present inventors have addressed the problem of providing aneffective formulation for the skin in which the active ingredients arederived from natural products.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow.

SUMMARY OF THE INVENTION

Embodiments of the invention are directed to methods of preparing aserum composition for application to skin which include one or more ofthe following steps:

(1) incubating eggs to form an embryoblast including an embryo andamniotic fluid,

(2) extracting amniotic fluid from the eggs,

(3) extracting the embryo or a portion of the embryo,

(4) homogenizing the embryo,

(5) centrifuging the embryo to obtain an embryo supernatant and anembryo pellet, and

(6) mixing the amniotic fluid from step (2) with the embryo supernatantand optionally the embryo pellet from step (5) at a ratio of 70-100%amniotic fluid and 0-30% embryo supernatant to provide the serumcomposition.

In preferred embodiments, the egg is a chicken egg and the incubationperiod is 5-14 days, more preferably the incubation period is 7-8 days.The serum composition may be used directly or combined with othercomponents. In some preferred embodiments, proteins, peptides, lipids,vitamins, minerals, moisturizers, plant extracts and/or preservativesare added to the serum composition.

Embodiments of the invention are directed to a serum composition whichincludes amniotic fluid and homogenized embryo mixed at a ratio of70-100% amniotic fluid with 0-30% homogenized embryo, obtained from achicken egg incubated for a period of 5-14 days after fertilization,preferably, the incubation period is 7-8 days.

Preferred embodiments of the invention are directed to formulations thatcontain the serum composition described above in combination with one ormore of the following: polyethylene glycol (PEG), hyaluronic acid,glycerin, soy protein, silk protein, ginko biloba, green tea extract,grape seed oil, rye seed extract, argireline, gluconodeltaloactone andsodium benzoate.

In preferred embodiments, serum compositions and formulations containingthe serum composition according to the invention are used in a method ofimproving the appearance of skin by applying the serum composition orformulation in an effective amount to an individual in need thereof. Inpreferred embodiments, the formulation is applied to the face, neckand/or hands. Preferably, the formulation is applied 1-3 times per day.

In preferred embodiments, compositions and formulations containing theserum composition according to the invention are used in a method oftreating burns by applying the serum composition or a formulationcontaining the serum composition in an effective amount to an individualin need thereof.

In preferred embodiments, serum compositions and formulations containingthe serum composition according to the invention are used in a method oftreating a scar or skin lesion by applying the serum composition or aformulation containing the serum composition in an effective amount toan individual in need thereof

In preferred embodiments, serum compositions and formulations containingthe serum composition according to the invention are used in a method oftreating wounds by applying the serum composition or a formulationcontaining the serum composition in an effective amount to an individualin need thereof. In some embodiments, the wound is a diabetic wound.

In preferred embodiments, the serum composition or formulationcontaining the serum composition is applied 1-3 times per day.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 shows C₂C₁₂ cells seeded at a density of 5000 cells per well in a96 well plate and cultured for 48 hours with DMEM medium under theindicated condition (n=12). The assay was performed as described byINVITROGEN's MTT Cell Proliferation Kit protocol. Extract X2 was addedat a concentration of 0-10% in the presence of fetal calf serum (FCS).

FIG. 2 shows the results of a study using a formulation containingExtract X2. A total of 32 females from age 35 to 60 years oldparticipated in this study. Dark bars indicate satisfied responses.Lighter bars indicate neutral responses. There were no unsatisfiedresponses in any of the tested categories.

FIG. 3 shows C57BL mice which were introduced with controlled burninjury and treated twice a day with specially formulated serumcontaining Extract X2. A significant speedy recovery was observed indays 1-7 (p<0.05) compared to control (PBS). For each condition, n=4;p<0.05 (compared to control).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

While the described embodiment represents the preferred embodiment ofthe present invention, it is to be understood that modifications willoccur to those skilled in the art without departing from the spirit ofthe invention. The scope of the invention is therefore to be determinedsolely by the appended claims.

As used herein, the terms “treating,” “treatment,” “therapeutic,” or“therapy” do not necessarily mean total cure or abolition of the diseaseor condition. Any alleviation of any undesired signs or symptoms of adisease or condition, to any extent can be considered treatment and/ortherapy. Furthermore, treatment may include acts that may worsen thepatient's overall feeling of well-being or appearance.

Embodiments of the invention are directed toward treating patients withskin diseases, undesirable skin conditions or appearances and towardtreating older skin to provide a younger appearance, i.e., preventing,inhibiting or relieving the effects of aging on skin and therebyimproving the appearance of wrinkled, lined, dry, flaky, aged or photodamaged skin and improving skin thickness, elasticity, flexibilityand/or plumpness at one or more particular sites. Preferred embodimentsof the invention are directed to treatment of wounds including diabeticwounds, scars, skin lesions and burns. More specifically, “treatment” isintended to mean providing a therapeutically detectable and beneficialeffect on a patient suffering from a skin condition or appearance whichthe patient has found to be undesirable.

The term “effective amount” is used to indicate an amount of an activecompound that elicits the biological or medicinal response indicated.This response may occur in a tissue, system, animal or human andincludes alleviation of the symptoms of the condition or disease beingtreated.

Embodiments of the present invention are directed to serum compositionsand formulations containing the serum compositions for treatment ofsymptoms associated with aging of the skin, treatment of dermatologicaldiseases, scars, burns, wounds, diabetic wounds and injuries related tothe skin. The present inventors have discovered natural active compoundsthat support health of skin cells. These gentle and effective compoundsare extracted from Epiblast-derived Stem Cells that are rich in naturalgrowth factors, amino acids, polypeptides, multi-vitamins, and mineralswhich could stimulate skin cell regeneration. The present inventorscombined these Epiblast-derived Stem Cell extracts with naturalExtra-Cellular Fluid (ECF), thereby creating a gentle but advancedAnti-Aging factor, “Extract X2”.

During embryogenesis, a fertilized egg divides (cleavage), forms a ballof cells (morula), develops a cavity (blastocyst stage), and forms thethree primary germ layers of cells that will ultimately give rise to allthe cell types of the body (gastrula stage), and ultimately generatesall the specialized tissues and organs of a mature organism. Theepiblast is a tissue type derived either from the inner cell mass inmammals or the blastodisc in birds and reptiles that becomes the embryo.The growth rate of cell mass during early embryogenesis is the fastestgrowth period in the whole life of any animal, showing linear growth. Inorder to support such a rapid growth, early embryonic cells must producea high level of growth promoting elements, and at the same timesufficient nutritional factors must be present in the environment.Embodiments of the invention are directed to extraction of these growthand nutritional elements for preparation of a formulation forapplication to skin. Without intending to be limited by theory, it isthought that the serum according to embodiments of the invention is ableto fool the skin cells to act like younger cells in growth phase. Asmost, if not all, growth factors were initially discovered in studyingembryogenesis, the growth factors in extracts described herein aregenerally present at biologic levels. Even though the level of any givengrowth factor may be low compared to a chemically synthesizedformulation, the combination of factors found in serum composition andformulations containing the serum compositions according to embodimentsof the invention could act synergistically. In addition, otherregulatory elements, such as peptides and cytokines, are also present.

An incomplete list of growth factors involved in embryogenesis is thefollowing: Adrenomedullin (AM); Anterior chamber growth factor;Autocrine growth factor; Basic fibroblast growth factor (FGF2); Bonemorphogenetic protein (BMP); Bone morphogenetic protein 2 (BMP2); Bonemorphogenetic protein 3 (BMP3); Bone morphogenetic protein 4 (BMP4);Bone morphogenetic protein 7 (BMP7); Bone morphogenetic protein 8B(BMP8B); Brain-derived neurotrophic factor (BDNF); Connective tissuegrowth factor (CTGF); Epidermal growth factor (EGF); Erythropoietin(EPO); Fibroblast growth factor (FGF); Fibroblast growth factor 1(FGF1); Fibroblast growth factor 3 (FGF3); Fibroblast growth factor 4(FGF4); Fibroblast growth factor 5 (FGF5); Fibroblast growth factor 7(FGF7); Fibroblast growth factor 8 (FGF8); Glial growth factor (GGF);Granulocyte colon-stimulating factor (G-CSF); Granulocyte macrophagecolon-stimulating factor (GM-CSF); Growth differentiation factor-9(GDF9); Hepatocyte growth factor (HGF); Hepatoma-derived growth factor(HDGF); Insulin-like growth factor (IGF-I); Insulin-like growth factorII (IGF-II); Interleukin 3 (IL-3); Interleukin 6 (IL-6); Keratinocytegrowth factor (KGF); Migration-stimulating factor; Myostatin (GDF-8);Nerve growth factor (NGF); Placental growth factor (PlGF);Platelet-derived growth factor (PDGF); Thrombopoietin (TPO);Transforming growth factor-α (TGF-α); Transforming growth factor-β(TGF-β); Transforming growth factor-β1 (TGF-β1); Transforming growthfactor-β2 (TGF-β2); Transforming growth factor-β3 (TGF-β3); vascularendothelial growth factors (VEGF); and Wnt protein family.

The following minerals are involved in embryogenesis: Sodium, Potassium,Chloride, Calcium, Phosphate, Magnesium, and trace metals (Iron, Copper,Zinc, Manganese, Selenium, and Molybdenum), etc.

The following vitamins are involved in embryogenesis: Vitamin A, VitaminB6, Folic acid, Vitamin B9, Vitamin B12, Vitamin C, Vitamin D3, andVitamin E, etc.

The following additional biochemical components are involved inembryogenesis: Glucose, Cholesterol, Triglyceride, Urea, Albumin,Globulin, Bicarbonate,

Oligo-peptides, CoQ10, Carnitine, Alpha-fetoprotein, SuperoxideDismutase, DNA, and RNA, etc.

Serum compositions according to the invention are termed “Extract X2”.As the Extract X2 composition according to embodiments of the inventionis obtained from embryonic cells at an early time of development, mostif not all of the above components are present in the Extract X2composition and formulations containing the Extract X2 composition.

Formulations containing an Extract X2 composition can be used to treatsymptoms of skin aging including but not limited to improvement in skinfirmness, whitening, moisturizing, dark spot reducing, wrinkle reducing,gloss, and elasticity. Formulations according to embodiments of theinvention are also useful to treat a variety of skin conditionsincluding but not limited to scars, burns, and wounds including diabeticwounds.

In some preferred embodiments, a blend of hyaluronic acid, soy proteinsand/or silk proteins helps strengthen and tighten the junction betweencells to lock in moisture, strengthening the lipid barrier andpreventing transepidermal water loss. In some preferred embodiments,antioxidants, such as green tea, ginkgo biloba extracts and/or grapeseed oil establish a powerful anti-radical defense system, shieldagainst pollution and skin stressors. In some preferred embodiments, ryeseed extract and/or oligo-peptide are added. The Extract X2 serumsoothes facial wrinkles, tightens skin and reveals moist delicate skin.Compositions according to embodiments of the invention reduce theappearance of fine lines and wrinkles, brighten skin tone, and leaveskin feeling soft, smooth, elastic, and firm.

Preparation of the Formulation

The starting material is the inner cell mass or epiblast (blastodisc inmammals and birds) derived from the fertilized eggs of birds, reptiles,amphibians or fish including but not limited to chickens, ostrich,turkeys, geese, ducks, and turtles. The cells of the inner cell mass arethe cells from which the embryo will develop. In a most preferredembodiment, the inner cell mass from fertilized chicken eggs is used asthe starting material. The eggs are incubated 5-14 days, preferable 7-8days after fertilization. After the incubation period, the eggs areopened and the amniotic fluid is extracted by any means known in theart. Typically, the amniotic fluid is simply extracted by means of asyringe from the amniotic cavity. The extracted amniotic fluid iscentrifuged to remove cells and debris and can be used immediately orstored at −20° C., preferably at −80° C.

The embryo portion from the epiblast is washed, preferably withdeionized water or buffered solution such as phosphate buffered saline.The embryos are homogenized and centrifuged to separate largerparticles. The supernatant is removed and may be used immediately orstored at −20° C., preferably at −80° C. The pellet, which is useful inheavier formulations such as creams, can be used immediately orlyophilized for future use.

In preferred embodiments, the final composition contains 50-100%amniotic fluid and 0-50% extracted embryos. So the formulation may havea ratio from 1:1 to 100:0 amniotic fluid: extracted embryos. In someembodiments, the composition may contain substantially only amnioticfluid. Preferably the final composition contains 70-100% of amnioticfluid and 0-30% of embryos. In preferred embodiments, the range is 70:30to 100:0 amniotic fluid: extracted embryos. In preferred embodiments,ratios of 100:0, 95:5, 90:10, 85:15, 80:20, 75:25, and 70:30 amnioticfluid:extracted embryos may be used.

In preferred embodiments, polyethylene glycol 150 Distearate (PEG 150Distearate) is added to protect large molecules and improve the feel ofthe formulation on skin. PEG is added at a concentration of 0.5-5%(w/v), preferably 0.5-2% (w/v), most preferably around 1% (w/v). Inpreferred embodiments, any PEG of molecular weight range 4,000-15,000may be used. In some embodiments, large molecular weight proteins, suchas bovine serum albumin or soy protein, may be added to the composition.In some embodiments, glyceryl monostearate may be substituted for PEG orused in addition to PEG.

In preferred embodiments, moisturizers may be added to the finalformulation. Preferably hyaluronic acid is added at a concentration of0.1-5% (w/v), preferably, 0.2-2% (w/v), most preferably about 0.5%(w/v). In some embodiments, glycerin, preferably vegetable-derived, isadded at a concentration of 2-10% (v/v), preferably, 5-7% (v/v), mostpreferably about 6% (v/v). Proteins, preferably hydrolyzed proteins, maybe added including but not limited to soy protein and silk protein.These proteins are added in a preferred concentration range of 0.5-5%,preferably 1-2%.

In preferred embodiments, the formulation may include antioxidant plantextracts such as Ginko Biloba at a concentration of 1-5% (v/v),preferably 2-4% (v/v), more preferably about 3% (v/v). Preferably, theformulation may contain green tea extract at a concentration of 1-5%(v/v), preferably 1-3% (v/v), more preferably about 2% (v/v).Preferably, the formulation may contain grape seed oil at aconcentration of 1-5% (v/v), preferably 1-3% (v/v), more preferablyabout 2% (v/v). In some embodiments, the formulation may include one ormore selected from extracts from citrus, olive tree, rosemary, sage,thyme, chamomile, berries, fruits, vegetables, herbs, cereals, treematerials, plant sprouts, and seeds. In some embodiments, theformulation may include one or more selected from vitamin A, thiamine,niacinamide, pyridoxine, riboflavin, cyanocobalamin, biotin, pantothenicacid, vitamin C, vitamin D, vitamin E, vitamin K and folic acid.

In preferred embodiments, the formulation may contain compounds whichreduce the degree or appearance of existing wrinkles such as rye seedextract at a concentration of 1-5% (v/v), preferably 2-4% (v/v), morepreferably about 3% (v/v) and/or extra oligo-peptides such as argirelineat a preferred concentration of 0.5-2% (v/v), preferably about 1% (v/v).In some embodiments, a formulation according to the invention maycontain one or more selected from extracts from cacao beans, cola nuts,and caffeine, theobromine, and theophylline, coffee berry, aloe, greentea, centella asiatica, Coenzyme Q10, and the like.

In preferred embodiments, the composition contains a preservative.Preferably, the preservative is one that is accepted by EOCERT as anacceptable preservative in certified organic cosmetics such asgluconodeltalactone and/or sodium benzoate. Preferably, theconcentration is 1-3% (w/v), more preferably about 1.5% (w/v) in total.Alternative preservatives which may be used include parabens (methyl-,ethyl-, propyl- and butyl-), urea derivatives such as imidazolidinylurea and diazolidinyl urea, isothiazolones (methylchloro-,methyl-isothiazolinone), halogen organic actives (iodopropynylbutylcarbamate, methyl-dibromo glutaronitrile), organic acids,chloracetamine, EDTA, phenoxyethanol, triclosan, DMDM-hydantoin andquaternium-15. In some preferred embodiments, the preservative isselected from natural preservatives such as extracts of willow bark,radish root ferment filtrate, grapefruit seed, extracts of rosemary,essential oils such as tea tree, neem seed and thyme and vitamins E orC.

In preferred embodiments, the components of the formulation are approvedfor use in certified organic cosmetics.

Embodiments of the skin formulation can contain optional ingredientsused commonly in external preparations for the skin. Preferred examplesof such optional ingredients include: oils/waxes such as macadamia nutoil, avocado oil, corn oil, olive oil, rapeseed oil, sesame oil, castoroil, safflower oil, cottonseed oil, jojoba oil, coconut oil, palm oil,liquid lanolin, cured coconut oil, cured oil, Japan wax, cured castoroil, beeswax, candelilla wax, carnauba wax, ibota wax, lanolin, reducedlanolin, hard lanolin, and jojoba wax; hydrocarbons such as liquidparaffin, squalane, pristane, ozokerite, paraffin, ceresin, vaseline,and microcrystalline wax; higher fatty acids such as oleic acid,isostearic acid, lauric acid, myristic acid, palmitic acid, stearicacid, behenic acid, and undecylenic acid; higher alcohols such as cetylalcohol, stearyl alcohol, isostearyl alcohol, behenyl alcohol,octyldodecanol, myristyl alcohol, and cetostearyl alcohol; syntheticester oils such as cetyl isooctanoate, isopropyl myristate, hexyldecylisostearate, diisopropyl adipate, di-2-ethylhexyl sebacate, cetyllactate, diisostearyl malate, ethylene glycol di-2-ethyl hexanoate,neopentylglycol dicaprate, glyceryl di-2-heptylundecanoate, glyceryltri-2-ethylhexanoate, trimethylolpropane tri-2-ethylhexanoate,trimethylolpropane triisostearate, and pentaerythritoltetra-2-ethylhexonate; silicone oil, such as chain polysiloxanes such asdimethylpolysiloxane, methylphenylpolysiloxane, anddiphenylpolysiloxane; cyclic polysiloxanes such asoctamethylcyclotetrasiloxane, decamethylcyclopentasiloxane, anddodecamethylcyclohexanesiloxane; modified polysiloxanes such asamino-modified polysiloxane, polyether-modified polysiloxane,alkyl-modified polysiloxane, and fluorine-modified polysiloxane; anionicsurfactants such as fatty acid soaps (such as sodium laurate and sodiumpalmitate), potassium laurylsulfate, and triethanolamine alkylsulfateether; cationic surfactants such as trimethyl ammonium stearyl chloride,benzalkonium chloride, and laurylamine oxide; amphoteric surfactantssuch as imidazoline-based amphoteric surfactants (such as a2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt),betaine-based surfactants (such as alkyl betaine, amide betaine, andsulfo betaine), and acylmethyl taurine; nonionic surfactants such assorbitan fatty acid esters (such as sorbitan monostearate and sorbitansesquioleate), glycerin fatty acid esters (such as glycerinmonostearate), propyleneglycol fatty acid esters (such aspropyleneglycol monostearate), cured castor oil derivatives, glycerolalkyl ether, POE sorbitan fatty acid esters (such as POE sorbitanmonooleate and polyoxyethylene sorbitan monostearate), POE sorbitolfatty acid esters (such as POE-sorbitol monolaurate), POE glycerol fattyacid esters (such as POE-glyceryl monoisostearate), POE fatty acidesters (such as polyethyleneglycol monooleate and POE distearate), POEalkyl ethers (such as POE2-octyldodecyl ether), POE alkylphenyl ethers(such as POE nonylphenyl ether), pluronic types, POE/POP alkyl ethers(such as POE/POP2-decyltetradecyl ether), tetronic types, POE castoroil/cured castor oil derivatives (such as POE castor oil and POE curedcastor oil), sucrose fatty acid ester, and alkyl glycoside; polyvalentalcohols such as polyethylene glycol, glycerin, 1,3-butylene glycol,erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropyleneglycoi, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4-hexanediol,1,2-hexanediol, and 1,2-octanediol; moisture components such as sodiumpyrrolidone carboxylate, lactate, and sodium lactate; fine particlessuch as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesiumcarbonate, silicic anhydride (silica), aluminum oxide, and bariumsulfate, whose surfaces may be treated; inorganic pigments such as rediron oxide, yellow iron oxide, black iron oxide, cobalt oxide,ultramarine blue, iron blue, titanium oxide, and zinc oxide, whosesurfaces may be treated; pearl agents such as mica titanium, fish scalefoil, and bismuth oxychloride, whose surfaces may be treated; organicdyes such as Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, BlueNo. 404, Yellow No. 5, Red No. 505, Red No. 230, Red No. 223, Orange No.201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No.201, Purple No. 201, and Red No. 204; organic fine particles such aspolyethylene powder, polymethyl methacrylate, nylon powder, andorganopolysiloxane elastomer; p-aminobenzoate-based ultravioletabsorbent; an anthranilate-based ultraviolet absorbent; asalicylate-based ultraviolet absorbent; a cinnamate-based ultravioletabsorbent; a benzophenone-based ultraviolet absorbent; a sugar-basedultraviolet absorbent; ultraviolet absorbents such as2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole, and4-methoxy-4′-t-butyldibenzoylmethane; lower alcohols such as ethanol andisopropanol; vitamins such as vitamin A or derivatives thereof; vitaminB types such as vitamin B₆ hydrochloride, vitamin B₆ tripalmitate,vitamin B₆ dioctanoate, vitamin B₂ or derivatives thereof, vitamin B₁₂,and vitamin B₁₅ or derivatives thereof; vitamin E types such asα-tocopherol, β-tocopherol, γ-tocopherol, and vitamin E acetate, vitaminD types, vitamin H, pantothenic acid, pantethine, and pyrroloquinolinequinone; and antibacterial agents such as phenoxyethanol.

Embodiments of the invention directed to external application forpromoting wound healing may preferably contain ingredients effective tothe regeneration of the wounded dermal tissue, including but not limitedto an antibiotic substance for preventing microbial infection such aspenicillin, fradiomycin, tetracycline, or a salt thereof, a sterilizersuch as acrinol or isodine, or the like. In more preferred embodimentsnatural extracts are used such as Aspen Bark Extract, Leuconostoc/RadishRoot Ferment Filtrate, Lavender Extract, Lemon Peel Extract, ThymeExtract, Goldenseal Extract, Echinacea Extract, and Hypericum Extract.

Embodiments of the invention directed to external application forpromoting healing of burns may preferably contain additional ingredientseffective to treat, heal or relieve pain from burn injuries such asantimicrobials including but not limited to bacitracin, silversulfadiazine, mafenide, silver nitrate, and povidone-iodine, antibioticsincluding but not limited to oxacillin, mezlocillin and gentamicin, asterilizer such as acrinol or isodine, and topical pain medications.

Embodiments of the skin formulation may be in any form suitable forretaining the formulation on the skin for a sufficient period of time,such as a serum, lotion, emulsion, ointment, or cream. In someembodiments, the skin formulation may be used in a cosmetic composition.

Administration may be to any part of the body. However, in preferredembodiments directed to anti-aging formulations, administration ispreferably to exposed skin such as face, neck and hands. The formulationmay be administered to all or part of a given area such as the face. Inpreferred embodiments directed to a particular dermatological disorderor injury such as a wound or burn, administration is to the affectedarea.

Administration may be 1 or more times daily until sufficient relief fromsymptoms is obtained. Preferably, administration is from 1-5 timesdaily, more preferably, 1-2 times daily. In some embodiments, once dailyadministration is sufficient. Administration may be continual for aperiod of time until symptoms are relieved or eliminated or intermittentif the patient suffers from recurring symptoms.

EXAMPLES Example 1 Method of Preparation of Serum Extract X2

Fertilized chicken eggs were incubated in an avian egg incubator at 100°C. for 8 days with automatic turning. The incubated eggs were opened andamniotic fluid is extracted by a needle. The amniotic fluid wascentrifuged at 4,000 rpm for 10 minutes. The supernatant was removed.

Embryos were washed with deionized H₂O or phosphate buffered saline(PBS). The embryos or a portion of the embryos were homogenized and thencentrifuged at 6,000 rpm for 15 minutes. The supernatant was removed.The pellet was dried by lyophilization for future use.

The extracted amniotic fluid and embryos were mixed at ratio from 70% to100% of amniotic fluid with 30% to 0% of extracted embryos, depending onthe final product.

Example 2 Effect of Serum Extract X2 on Cell Growth

FIG. 1 shows C₂C₁₂ cells seeded at densities of 5000 cells per well in a96 well plate and cultured for 48 hours with DMEM medium under theindicated condition (n=12). The assay was performed according toINVITROGEN's MTT Cell Proliferation Kit protocol. Serum Extract X2 at aratio of amniotic fluid:extracted embryos of 100:0 were added at aconcentration of 0-10% in the presence of fetal calf serum (FCS). Theresults suggest that serum Extract X2 has sufficient nutritionalsupplements to replace FCS in cell culture (light blue bars in thefigure). More important, serum Extract X2 synergistically promoted cellproliferation with FCS (10% FCS vs. 10% FCS+10% X2, P<0.05).

Example 3 Method of Preparation of Formulation Containing Extract X2

1% (w/v) PEG, hyaluronic acid at 0.5% (w/v), vegetable glycerin at 6%(v/v), soy proteins at 2% (v/v), silk proteins at 1% (v/v), GinkgoBiloba at 3% (v/v), Green Tea extract at 2% (v/v), Grape Seed Oil at 2%(v/v), Rye Seed Extract at 3% (v/v), and extra oligo-peptide, such asArgireline at 1% (v/v) was added into the final formulation which had10% Extract X2.

Gluconodeltalactone and Sodium Benzoate, (both are accepted by ECOCERTas a preservatives for use in certified organic cosmetics), were addedat 1.5% (w/v) total into the final formulation.

TABLE 1 Exemplary formulation containing Extract X2 ComponentConcentration Extract X2 10% (v/v) PEG 150 Distearate 1% (w/v)hyaluronic Acid 0.5% (w/v) DL-Pathenol 1.0% (w/v) vegetable glycerin 6%(v/v) Quaternium-79 Hydrolyzed Soy Protein 2% (v/v) Hydrolyzed SilkProtein 1% (v/v) Ginkgo Biloba Leaf Extract 3% (v/v) Camellia SinensisLeaf (Green Tea) Extract 2% (v/v) Vitis Vinifera (Grape) Seed Oil 2%(v/v) Secale Cereale (Rye) Seed Extract 3% (v/v) Acetyl Hexapeptide-8(Argireline) 1% (v/v) Gluconodeltalactone and Sodium Benzoate 1.5% (w/v)water 67% (v/v)

Example 4 Effect of Extract X2 Formulation on Skin

A formulation as in Table 1 above was tested in 32 females aged 35-60years old for 30 days. A number of criteria were evaluated includingskin firmness, whitening, moisturizing, dark spot reducing, wrinklereducing, gloss, nourishing, and elasticity. Texture, maintenance(stability), absorbance and distribution of the product on skin werealso evaluated. The results are shown in FIG. 2. In all categories, theresults were positive. For overall performance, 80% were satisfied withthe formulation. No side effects were reported in this study. Noindividuals were unsatisfied with the results after 30 days of testing.

Example 5 Effect of Extract X2 Formulation on Burn Injury

Anesthetized male C57BL mice were introduced with controlled burn injuryand treated twice a day with specially formulated serum as described inTable 2 below containing Extract X2. A significant speedy recovery wasobserved in days 1-7 (p<0.05) compared to control (PBS) (FIG. 3).

TABLE 2 Exemplary formulation for burn injury containing Extract X2Component Concentration Extract X2 10% (v/v) Aloe Vera 200x 0.4% (w/v)hyaluronic Acid 1.0% (w/v) DL-Pathenol 1.0% (w/v) vegetable glycerin 6%(v/v) Quaternium-79 Hydrolyzed Soy Protein 2% (v/v) Organicals AcneExtract 4% (v/v) Organicals Tissue Repair 4% (v/v) Niacinamide 2% (v/v)Vitamin C 2% (v/v) Willow Bark Extract 2.5% (v/v) Camellia Sinensis Leaf(Green Tea) Extract 2% (v/v) Yeast Extract 2% (v/v) Vitis Vinifera(Grape) Seed Oil 2% (v/v) Hydrocotyl (Centella Asiatica) Extract 3%(v/v) Glycrrhiza Glabra (Licorice) Root Extract 2% (v/v) Olea Europaea(Olive) Leaf Extract 2.0% (v/v) Leuconostoc/Radish Root Ferment Filtrate2.0% (w/v) water 50.1% (v/v)

Example 6 Effect of Extract X2 Formulation on Wounds

A formulation as in Example 3 above is prepared except that Extract X2is prepared at a ratio of amniotic fluid:extracted embryos of 90:10 andthe formulation further contains at least one of Aspen Bark Extract,Lavender Extract, Lemon Peel Extract, Thyme Extract, Goldenseal Extract,Echinacea Extract, or Hypericum Extract. The formulation is applied 2times a day to a skin wound.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

What is claimed is:
 1. A method of making a serum composition forapplication to the skin of a human in need thereof consistingessentially of: a) incubating chicken eggs to form an embryoblastcontaining an embryo and amniotic fluid for 5-14 days; b) extractingamniotic fluid from the eggs; c) extracting the embryo from the eggs; d)homogenizing the embryo or a portion thereof; e) centrifuging the embryoto obtain an embryo supernatant and an embryo pellet; f) mixing theamniotic fluid from step (b) with the embryo supernatant and the embryopellet from step (e) at a ratio of 70-100% amniotic fluid and 0-30%embryo supernatant to provide an amniotic fluid/embryosupernatant/pellet; g) adding at least one component selected from thegroup consisting of polyethylene glycol, hyaluronic acid, glycerin, soyprotein, silk protein, ginko biloba, green tea extract, grape seed oil,rye seed extract, argireline, gluconodeltalactone, and sodium benzoateto said amniotic fluid/embryo supernatant/pellet to form said serumcomposition; h) applying the serum composition to the skin of the humanin need thereof.
 2. The method of claim 1, wherein the incubation periodin step (a) is 7-8 days.